Population Genetic Structuring in Opisthorchis viverrini over Various Spatial Scales in Thailand and Lao PDR Nonglak Laoprom 1,2 , Paiboon Sithithaworn 1,2 *, Ross H. Andrews 1,3 , Katsuhiko Ando 4 , Thewarach Laha 1 , Sirawut Klinbunga 5 , Joanne P. Webster 6 , Trevor N. Petney 7 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 2 Liver Fluke and Cholangiocarcinoma Research Center (LFCRC), Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 3 Faculty of Medicine, Imperial Collage London, London, United Kingdom, 4 Department of Medical Zoology, School of Medicine, Mie University, Tsu, Japan, 5 National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand, 6 Department of Infectious Disease Epidemiology, School of Public Health, Faculty of Medicine, Imperial Collage London, London, United Kingdom, 7 Institute of Zoology 1: Ecology and Parasitology, Karlsruhe Institute of Technology, Karlsruhe, Germany Abstract Khon Kaen Province in northeast Thailand is known as a hot spot for opisthorchiasis in Southeast Asia. Preliminary allozyme and mitochondrial DNA haplotype data from within one endemic district in this Province (Ban Phai), indicated substantial genetic variability within Opisthorchis viverrini. Here, we used microsatellite DNA analyses to examine the genetic diversity and population structure of O. viverrini from four geographically close localities in Khon Kaen Province. Genotyping based on 12 microsatellite loci yielded a mean number of alleles per locus that ranged from 2.83 to 3.7 with an expected heterozygosity in Hardy–Weinberg equilibrium of 0.44–0.56. Assessment of population structure by pairwise F ST analysis showed inter-population differentiation (P,0.05) which indicates population substructuring between these localities. Unique alleles were found in three of four localities with the highest number observed per locality being three. Our results highlight the existence of genetic diversity and population substructuring in O. viverrini over a small spatial scale which is similar to that found at a larger scale. This provides the basis for the investigation of the role of parasite genetic diversity and differentiation in transmission dynamics and control of O. viverrini. Citation: Laoprom N, Sithithaworn P, Andrews RH, Ando K, Laha T, et al. (2012) Population Genetic Structuring in Opisthorchis viverrini over Various Spatial Scales in Thailand and Lao PDR. PLoS Negl Trop Dis 6(11): e1906. doi:10.1371/journal.pntd.0001906 Editor: David Blair, James Cook University, Australia Received November 13, 2011; Accepted October 2, 2012; Published November 15, 2012 Copyright: ß 2012 Laoprom et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through health cluster (SHeP-GMS), Khon Kaen University (grant number: H-2553-PD-2, www.nru.go.th), the Thailand Research Fund Basic Research Grant (grant number: BRG5280021, www.trf.or.th), the Invitation Research Grant Faculty of Medicine Khon Kaen University (grant number: I52227, www. md.kku.ac.th) and by the German Federal Research Foundation (grant number: DFG:PE1611/3-1, www.dfg.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: paib_sit@hotmail.com Introduction The liver fluke, Opisthorchis viverrini is a food-borne trematode endemic in Southeast Asia, including Thailand, Lao PDR, Vietnam and Cambodia with more than 10 million people infected [1,2,3]. Infection occurs by eating raw or uncooked cyprinid fish containing metacercariae [4,5]. O. viverrini infection is a significant medical problem because of its involvement as a major risk factor causing bile duct cancer (cholangiocarcinoma, CCA) [6]. Liver cancer, predominantly CCA, is the fourth and fifth cause of mortality in males and females, respectively in Thailand [7]. Globally, Khon Kaen Province, Thailand is one of the hot spots of CCA with incidence levels (per 100,000) of 78.4 in males and 33.3 in females [8]. Recently, we reported that O. viverrini does not represent a single species but consists of at least two morphologically similar but genetically distinct (i.e. cryptic) species from Thailand and Lao PDR [9]. We also showed that there were at least six genetically distinct groups that are associated with different major wetlands. Additionally, biological variation between populations of O. viverrini from different wetlands in Thailand and Lao PDR has been detected. For instance, worm recovery as well as the fecundity of O. viverrini from the Songkram River in Thailand was significantly different from other wetland systems (Chi, Mun and Wang Rivers) in Thailand and Lao PDR (Nam Ngum River) [10]. Furthermore, worms belonging to this population were signifi- cantly different in body size from populations from the Chi and Nam Ngum River wetlands [10]. The fine scale population genetics of O. viverrini has to date only been studied from a single locality (Ban Phai in Khon Kaen, Thailand), but the results indicated considerable genetic diversity and heterozygote defi- ciency occurring within a small geographical area [11]. More detailed information on the population genetic structure of O. viverrini is, however, needed to fully determine whether population substructuring and/or differential genetic diversity are associated with geographical differences in distinct wetlands, river systems and flooding patterns [12]. Recently, we characterized, optimized and demonstrated the utility of microsatellite DNA markers for O. viverrini and provided evidence of population subdivision over a large spatial scale with the maximum distant apart of up to 770 km [13]. However, whether such a population pattern occurs over a small spatial scale or not is unknown. In this study, we examined the genetic diversity and population structure of O. viverrini populations occurring within and between PLOS Neglected Tropical Diseases | www.plosntds.org 1 November 2012 | Volume 6 | Issue 11 | e1906